Alkaline protease mutant with improved specific enzyme activity at low temperature and application

Abstract

The invention belongs to the field of microbial genetic engineering and protein engineering, and particularly relates to an alkaline protease mutant with improved specific enzyme activity at low temperature and application. The alkaline protease mutant with the specific enzyme activity increased to 4770U / mg from original 3975U / mg at the temperature of 25 DEG C is obtained by performing site-directed mutagenesis on the alkaline protease gene as shown in SEQ ID NO.1, and the method is simple and easy to implement and lays a foundation for efficient expression of alkaline protease by bacillus licheniformis, so that fermentation production of the alkaline protease is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic engineering and protein engineering transformation, and in particular relates to an alkaline protease mutant with improved specific enzyme activity at low temperature and an application thereof. Background Art

[0002] Alkaline protease (ALK) refers to a class of enzymes that can hydrolyze protein peptide bonds in the alkaline pH range. It has been widely used in the detergent, pharmaceutical, leather and food industries. Among them, ALK derived from Bacillus Clausii is mainly used in the detergent industry, but its activity at low temperatures is still limited, which limits the effect of detergents under low temperature conditions. Therefore, this patent aims to screen ALK mutants with high activity at low temperatures through molecular modification of ALK, in order to meet the current demand for low-temperature ALK in the washing industry. This patent determines the enzymatic activity of alkaline protease according to GB / T 23527-2009.

[0003] The principle of alkaline protease activity determination in GB / T 23527-2009 is: under certain temperature and pH conditions, protease hydrolyzes casein substrate to produce phenolic amino acids (such as tryptophan, tyrosine, etc.). Under alkaline conditions, Folin phenol reagent can be reduced to blue and can be detected at OD680. The absorbance of the solution is measured at OD680 using a spectrophotometer. The enzyme activity is proportional to the absorbance and the enzyme activity of the alkaline protease can be calculated. The national standard method is the most widely used method for determining the activity of alkaline protease, and its advantages are good repeatability and low detection cost.

[0004] The pH of detergent is generally between 9.0 and 11.0. Alkaline proteases are mainly used in the detergent industry due to their high stability and activity under alkaline conditions. Their use in detergent formulations accounts for 89% of their total sales. According to surveys, the temperature for washing clothes in my country is usually around 25°C. However, most of the proteases added to commercially available detergents are medium-temperature alkaline proteases, which cannot exert their high activity at low temperatures, which has a great impact on the washing performance of detergents. Based on the above background, the applicant has molecularly modified the reported alkaline protease (ALK) and screened for ALK mutants with high activity at low temperatures to meet the current needs of the washing industry. Summary of the invention

[0005] The purpose of the present invention is to provide an alkaline protease mutant with improved enzyme activity at low temperature. The amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide the use of the mutant in the preparation of alkaline protease.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant artificially synthesized the starting alkaline protease sequence (Li J, Jiang L, Cao X, et al. Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular dyna mics simulation [J]. Enzyme and Microbial Technology, 2021, 147: 109787-. DOI: 10.1016 / j.enzmictec.2021.109787.), as shown in SEQ ID NO.1, and then modified the alkaline protease enzyme by mutating the glycine at position 113 to proline (i.e., as shown in SEQ ID NO.3). The specific enzyme activity of the alkaline protease at 25°C was increased from the original 3975U / mg to 4770U / mg, and the specific activity was greatly improved.

[0009] The protection scope of the present invention also includes:

[0010] A fusion protein obtained by fusing the mutant protein described in SEQ ID NO.3 with a protein tag.

[0011] The gene encoding the mutant or fusion protein described in SEQ ID NO.3.

[0012] An expression cassette, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell having the above coding gene.

[0013] The mutant, fusion protein, mutant or fusion protein encoding gene of SEQ ID NO.4, expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the encoding gene are used in preparing metabolites.

[0014] A method for improving the enzymatic activity of alkaline protease comprises the following steps: performing the following mutation on the alkaline protease: mutating the 113th glycine G in SEQ ID NO.1 to proline P.

[0015] A method for preparing alkaline protease with improved specific enzyme activity comprises the following steps: preparing alkaline protease with improved specific enzyme activity by culturing the above-mentioned recombinant microorganism.

[0016] The encoding gene of the mutant described in SEQ ID NO.3 is preferably shown in SEQ ID NO.4.

[0017] In the above-mentioned uses or methods, preferably, the recombinant microorganism is a recombinant Bacillus licheniformis.

[0018] In the above-mentioned use or method, preferably, the Bacillus licheniformis is Bacillus licheniformis DW2.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The present invention performs site-directed mutagenesis on the alkaline protease gene shown in SEQ ID NO.1 to obtain an alkaline protease mutant with the specific enzyme activity at 25°C increased from the original 3975U / mg to 4770U / mg. The method is simple and easy to implement, lays a foundation for the efficient expression of alkaline protease by Bacillus licheniformis, and thus promotes the fermentation production of alkaline protease. DETAILED DESCRIPTION

[0021] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0022] Nickel affinity chromatography filler (Ni Sepharose 6Fast Flow) and molecular sieve filler (Sephadex G-25) were purchased from GE and loaded into the column according to the requirements of protein purification; the remaining reagents were domestically produced or imported analytical grade.

[0023] Embodiment 1:

[0024] Construction of alkaline protease mutants from Bacillus clausii

[0025] The artificially synthesized ALK gene is shown as SEQ ID NO.2, and the protein encoded by the gene is shown as SEQ ID NO.1.

[0026] The synthesized ALK gene was connected to the expression vector pHY-300 through the restriction sites NdeI and NotI to construct the recombinant plasmid pHY-300-ALK. The recombinant plasmid was transformed into Bacillus licheniformis DW2 (CN116656712A) to obtain recombinant Bacillus licheniformis expressing wild-type ALK. Recombinant Bacillus licheniformis pHY-300-ALK expressing the alkaline protease mutant shown in SEQ ID NO.1 G113P The specific preparation steps are as follows:

[0027] Using the plasmid pHY-300-ALK expressing the wild-type ALK gene as a template, the primers G113P-F and G113P-R were used to amplify the DNA fragment by PCR reaction. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min.

[0028] The above PCR products were separated by agarose gel electrophoresis to obtain the target DNA, purified by Winzyme's agarose gel recovery kit, and recombined and cyclized the DNA fragments using Winzyme's HI-Efficienct Cloning Mix, and then the recombined DNA was transformed into Escherichia coli DH5a competent cells. The bacteria were spread on a culture plate containing Tet resistance for screening and cultured in a 37°C incubator; the transformants were verified by colony PCR, and the primers used were amp-F and amp-R. If the target size was correct, the next step of sequencing could be performed. The vector nucleotide sequence determination was completed by Wuhan Jinkairui Bioengineering Co., Ltd.; the sequencing results were analyzed. If the sequence was consistent with the design, the mutant vector was successfully constructed. The recombinant plasmid of the mutant was extracted with a plasmid extraction kit for later use. The recombinant vector was transferred into the competent state of Bacillus licheniformis DW2. The bacteria were spread on a culture plate containing Tet resistance for screening and cultured in a 37°C incubator. The transformants were verified by colony PCR. The primers used were amp-F and amp-R. If the target size was correct, it was the engineering strain pHY-300-ALK. G113P Build successful.

[0029] The applicant also prepared other mutant proteins as a control group, and the recombinant strains expressing these mutant proteins (as shown in Table 1, including pHY-300-ALK S36A , pHY-300-ALK T51I , pHY-300-ALK H62L , pHY-300-ALK L94C , pHY-300-ALK I105V , pHY-300-ALK G113L , pHY-300-ALK G113T ) The construction process can refer to the recombinant Bacillus licheniformis pHY-300-ALK G113P The corresponding primers used in the construction of the recombinant strain are shown in Table 1.

[0030] Table 1 Primers used for site-directed mutagenesis

[0031]

[0032]

[0033] Embodiment 2:

[0034] Purification and activity determination of alkaline protease expressed by recombinant Bacillus licheniformis prepared in Example 1:

[0035] The wild-type ALK recombinant Bacillus licheniformis and mutant ALK recombinant Bacillus licheniformis prepared in Example 1 were streaked on a solid Tet plate (containing 20ug / mL of kanamycin) and cultured overnight at 37°C to obtain a single clone. Then pick a single clone and activate it in a liquid Tet medium (5mL), and culture it at 37°C for 12h. Then take 1ml of the activated bacterial solution and inoculate it in 20ml of liquid Tet medium for culture, and culture it at 37°C for overnight with shaking. The overnight cultured seed solution was inoculated into 20mL soybean meal medium at a 1% inoculum, fermented at 37°C and 230rpm for 48h, and the supernatant was collected by centrifugation. The supernatant was loaded onto a nickel affinity chromatography column, and the impurities were eluted with an equilibrium buffer (PBS, containing 20mM imidazole), and finally the target protein was eluted with an elution buffer (containing 300mM imidazole). The eluted protein solution was then desalted by molecular sieves to collect the purified AKL. The enzyme activities of wild-type ALK and 6 ALK mutants after purification at 25°C were measured respectively, and the specific enzyme activities of wild-type and mutants were calculated. The calculation formula of the change range is: (specific enzyme activity of mutants-specific enzyme activity of wild-type) / specific enzyme activity of wild-type.

[0036] The formula of the soybean meal culture medium includes: 45g / L corn starch, 50g / L soybean meal powder, 6g / L calcium carbonate, 5g / L ammonium sulfate, and natural pH.

[0037] The alkaline protease activity determination method refers to the Folin phenol method in Appendix B of GB / T 23527-2009, and the reaction temperature in this method is adjusted to 25°C. Enzyme activity definition: Under the conditions of pH 10.5 and temperature 25°C, the amount of enzyme required to catalyze the production of 1umol / L-tyrosine per minute is defined as 1 unit (U).

[0038] The results are shown in the following table:

[0039]

[0040]

[0041] From the above table, we can see that the wild-type AKL has an activity of 3975 U / mg at 25°C, and the AKL mutant G113P has a significantly improved enzyme activity, which is 4770 U / mg at 25°C, an increase of 20%.

Claims

1. An alkaline protease mutant, the amino acid sequence of the protease mutant is shown in SEQ ID NO.

3.

2. A fusion protein obtained by fusing the protease mutant according to claim 1 with a protein tag.

3. A gene encoding the protease mutant according to claim 1 or the fusion protein according to claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 3.

5. Use of the protease mutant according to claim 1, the fusion protein according to claim 2, the encoding gene according to claim 3, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell according to claim 4 in the preparation of alkaline protease.

6. A method for improving the activity of alkaline protease, comprising the following steps: mutating the glycine G at position 113 of SEQ ID NO.1 to proline P. .

7. A method for preparing alkaline protease with improved specific enzyme activity, comprising the following steps: preparing by culturing the recombinant microorganism according to claim 5.

8. The coding gene according to claim 3, wherein the coding gene of the mutant described in SEQ ID NO.3 is shown in SEQ ID NO.

4.

9. The use according to claim 5, wherein the recombinant microorganism is a recombinant Bacillus licheniformis.

10. The use according to claim 9, wherein the Bacillus licheniformis is Bacillus licheniformis DW2.

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